EP0822261B1

EP0822261B1 - Methods for rapid antimicrobial susceptibility testing

Info

Publication number: EP0822261B1
Application number: EP96305535A
Authority: EP
Inventors: Konan Peck; Pan-Chyr Yang; Shu-Li Wung
Original assignee: Academia Sinica
Current assignee: Academia Sinica
Priority date: 1996-07-29
Filing date: 1996-07-29
Publication date: 2002-07-03
Anticipated expiration: 2016-07-29
Also published as: EP0822261A1; JPH1066598A; US5789173A

Description

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to methods for rapidly identifying and quantitating efficacious antimicrobial agents.

2. Description of Prior Art

In recent years, the emergence of strains of drug-resistant bacteria has become a major health problem in many parts of the world. This has made it vitally necessary both to develop new antibacterial drugs and establish effective strategies to combact invading bacteria.
Presently, to treat patients afflicted with severe microbial infections, a random guessed broad spectrum antibiotics often fails to yield satisfactory results.
Antimicrobial susceptibility testing often be used to determine the right antimicrobial agents. It takes two to three days to complete the test, either by dilution method or by diffusion method. For frail or immuno-suppressed patients already in the process of developing septic shock, finding the right antibiotics in time is critical, because many of the patients could not survive the three-day period required by conventional antimicrobial susceptibility testing.
Traditional susceptibility testing methods rely on observation of bacterial colony formation, which requires prolonged incubation in order to accumulate sufficient mass to be visible. (See for example, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically" NCCLS Document M7-A2, 10(8), 2nd eds., 1990.)
There are many methods and instruments developed to identify bacterial pathogens with shorter time intervals; see for example, U.S.P. 4,385,115 issued to Hoffmann-La Roche Inc. N. J., U.S.A; U.S.P. 5,112,745 issued to Space Medical System, Inc., Garrett Park, MD; U.S.P. 5,089,395 issued to Univ. of Cincinnati, Cincinnati, OH; and U.S.P. 5,059,522 issued to L.G. Wayne, Irvine CA.
Nevertheless, pathogen identification does not necessarily provide information of what antibiotic drug yields pathogen susceptibility. For most of the nosocomial or community acquired bacterial infections, the pathogens are indigenous flora rather than epidemic pathogens that cause, for example, cholera or typhus and must be reported to government health administrations. For most physicians and patients, identification of the antimicrobial agent which can cure the illness is more important in therapeutics than knowing the nature of the pathogen itself.
In conventional antimicrobial susceptibility testing whether it is the micro-dilution or the disk diffusion method, primary culture of specimens on agar plates is required in order to observe the bacterial growth and identify the pathogen species. Subcultures are then performed to enrich bacterial population. For many types of conventional susceptibility testing, a standard concentration of bacterial inoculum must be used. The concentration of bacteria in a liquid medium can be determined in several ways including measurement of the optical density of a broth culture or by comparing the turbidity of a liquid medium to a standard representing a known number of bacteria in suspension. To test for susceptibility of pathogens to antimicrobial agents, the standardized inoculum is mixed with various antibiotics of different concentrations and the susceptibility is determined by visual examination of bacterial growth.
Newer methods using instruments to detect bacterial growth based on photometric detection of broth turbidity, radiometric detection of bacterial metabolites, or fluorometric detection of hydrolyzed fluorogenic substrates incorporated in culture medium (see e.g. C. Thornsberry et al., J. Clin Microbiol. 12, 375 (1980); F. S. Nolte et al., J. Clin. Microbiol. 26, 1079 (1988); J. L. Staneck et al., J. Clin. Microbiol. 22, 187 (1985); P. G. Beckwith et al., J. Clin. Microbiol. 15, 35 (1982)) have reduced the incubation time to a few hours.
Despite the improvements in instrumentation, all the prior art require primary culture and subculture routines which enrich bacterial concentrations to levels sufficiently high to be standardized by visual methods such as McFarland nephelometry. This inevitable procedure of inoculum concentration standardization is due to the fact that minimum inhibition concentration (MIC) of antibiotics is dependent on inoculum concentration in the prior art.
Superisingly, the invention has found that antimicrobial susceptibility testing can be conducted without the need of primary culture of specimen, or knowing the identity of the pathogen. Identification of the pathogen, however, can be done in parallel by conventional methods, if necessary.

SUMMERY OF THE INVENTION

It is an object of this invention to provide a rapid antimicrobial susceptibility testing method without primary culture process.
It is another object of this invention to perform antimicrobial susceptibility testing without prior species identification of microbes.
It is another object of this invention to determine MIC values without standardization of inoculum concentration.
It is another object of this invention to provide fast antimicrobial susceptibility testing for well known slow growing microorganisms such as fungi and mycobacteria.
It is a further object of this invention to rapidly determine MIC values by simultaneously detecting a large number of PCR products in minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention:
FIG. 1 shows the comparison between two conventional antimicrobial susceptibility testing methods and the invention in terms of steps and time consumed.
FIG. 2 shows MIC value vs. PCR cycle number and inoculum concentration to demonstrate that the MIC value is independent of inoculum concentration at low concentrations.
FIG. 3 shows the antimicrobial susceptibility testing results of four antibiotics to E. coli strain ATCC 25922: ampicillin (
); cephalothin (
); nalidixic acid (
); and gentamicin (
). The MIC values agree well with the values published by NCCLS.

DETAILED DESCRIPTION OF THE INVENTION

The invention in the first aspect provides a method for rapid antimicrobial susceptibility testing, which comprises:
(a) incubating a specimen; for example of human body fluids, blood, or others containing microbe(s), in a medium comprising an antimicrobial agent at serial dilution concentrations for from 1 to 6 hours to create differential microbial counts;
(b) amplifying the differential microbial counts by in vitro microbial DNA replication;
(c) contacting the amplified differential counts with one or more reporter molecules which are specific to double stranded DNA (dsDNA) and form complexes therewith; and
(d) observing the presence or absence of signal emission from the reporter molecule-dsDNA complexes.
Fig. 1 depicts the differences between two conventional antimicrobial susceptibility testing methods and the invention in terms of steps and time consumed. As clearly shown, the method in accordance with the invention avoids the primary culture process to incubate specimen directly in antibiotics embedded media without standardizing bacterial concentration. Also, the method in accordance with the invention rapidly identifies efficacious antimicrobial agents against pathogens without prior knowing the identity of the pathogens.
In a further aspect, the invention provides a method for rapid determination of minimum inhibitory concentration (MIC) values of antimicrobial agents which comprises:
(a) incubating specimens, for example of human body fluids, blood and others, comprising one or more microbes in a medium comprising an antimicrobial agent at serial dilution concentrations for from 1 to 6 hours to create differential microbial counts;
(b) amplifying the differential microbial counts by in vitro microbial DNA replication;
(c) contacting the amplified differential counts with one or more reporter molecules which are specific to dsDNA and form complexes therewith; and
(d) recording the lowest concentration of antimicrobial agent which does not result in signal emission from the reporter molecule-dsDNA complexes as the MIC value.
In accordance with the method of this invention, MIC values are dependent on bacterial concentration only at high bacterial concentrations. The system of the invention thus evades the lengthy primary culture procedures in an innovative way. This concept is further taught and validated by Figure 2 to show that the MIC values are independent of PCR amplification cycles and concentration of bacteria at low inoculation concentrations. At concentrations as high as 1 x 10⁶ CFU/ml which are about 1/100 of the inoculation concentration, 0.5 McFarland standard (1.5 X 10⁸ CFU/ml), used in prior art, the MIC values vary with inoculum concentration as shown in Figure 2.
To incubate specimens in antibiotics embedded media, any incubation containers conventionally used are suitable. In this regard, 96-well microtiter plates can be conveniently used. Each column of a 96-well plate contains culture medium (such as Mueller-Hinton broth) together with one antibiotic at various concentrations in eight different rows. Therefore, with one column reserved for controls, one 96-well microtiter plate can be used to test eleven different essential antibiotics at 8 different concentrations.
Contrary to the lengthy incubation period of the prior art, the incubation time of this invention can be significantly reduced. The suitable incubation time for bacteria in accordance with the invention can range from 1-12 hours. Preferably, it is within 1-6 hours. The most preferred incubation time is as short as three hours.
Taking Escherichia coli for example, its replication time is twenty minutes during logarithmic growth phase. In three hours, the bacterial count increases 512 times in the wells containing inefficacious antimicrobial agents. On the other hand, wells containing efficacious antimicrobial agents or effective concentrations would yield no bacterial growth.
For some fastidious bacteria such as Haemophilus species or N. gonorrhoeae, the Mueller-Hinton broth may require supplementation to support the growth of the bacteria. To compensate for these possible false negative results due to fastidious bacteria, a column of micro-wells may be reserved to contain DNA amplification primers specific to the fastidious bacteria species and medium supplements.
The method of invention is also applicable to susceptibility testing for slow growing microorganisms such as fungi or mycobacteria, for example, microorganisms with a replication time of longer than one hour.
Accordingly, the invention in another aspect provides a method for rapid antimicrobial susceptibility testing for slow growing microorganisms, which comprises:
(a) incubating a specimen comprising one or more slow-growing microorganisms in medium comprising an antimicrobial agent at serial dilution concentrations for less than 24 hours to create differential microbial counts or, when the slow-growing microorganism is Mycobacterium tuberculosis, for less than 10 days;
(b) amplifying the differential microbial counts by in vitro microbial DNA replication;
(c) contacting the amplified differential counts with one or more reporter molecules which are specific to double stranded DNA (dsDNA) and form complexes therewith; and
(d) observing the presence or absence of signal emission from the reporter molecule-dsDNA complexes.
In this application, antifungal agents such as Fluconazole, Nystatin, Amphotericin B, etc., are used instead of antibacterial agents for fungal antimicrobial susceptibility testing, and longer incubation time will be required due to the nature of the microorganisms. For antimicrobial susceptibility testing of the well known slow growing Mycobacterium tuberculosis, the incubation time is increased to 6 days and anti-tuberculosis drugs such as rifampin, isoniazid, and ethambutol are used instead.
The susceptibility testing results can be obtained in one to several days. Preferably, the results are obtained in one day for fungi, and less than 10 days for mycobacteria, as compared with the usual 6 to 8 weeks required by prior art.
Since this invention teaches revolutionary concepts to evade time consuming primary culture and species identification, in order to distinguish bacterial infection from fungal or mycobacterial infection when the results show resistance to all the antibacterial agents being tested, two micro-wells containing no antibiotics but DNA amplification primers specific to fungi and mycobacteria, respectively, are needed.
Specimens from different human systems also require different treatments. For example, specimens collected from sites of abscess or urinary tracts require dilution while blood or cerebrospinal fluid (CSF) specimens need additional two to three hour enrichment incubation to overcome the statistical thresholds.
In order to rapidly remove pathogens from the blood and its antibacterial properties together with antimicrobial agents that were applied to patients, lysis centrifugation by using an Isolator system (Wampole Lab, Cranbury, New Jersey) can be a suitable method. The blood cells are lysed in a glass tube and the content is centrifuged at 3,000 x g for 30 minutes to precipitate the pathogens.
Because the number of bacterial counts is typically very small in blood specimens, in order to avoid zero pathogens in any micro-well, the blood specimens need additional enrichment culture. To save time, breakpoint susceptibility testing rather than MIC test to determine whether the pathogen is susceptible, intermediate, or resistant to various antimicrobial agents may be more practical for patients in critical condition. In this manner, 20 micro-wells are sufficient to test 10 different antibiotics.
According to Poisson distribution calculation, in order to have less than 10% probability of having zero bacteria in any well, each well must have, on average, 3 bacterial counts. For most of the common bacterial pathogens, especially for those exist in normal flora, a three-hour enrichment incubation should be sufficient to gain enough bacterial counts to overcome the statistical barrier.
To test for antimicrobial susceptibility or to measure the MIC values, a short period of incubation in antimicrobial agent embedded medium creates necessary differential bacterial counts but the number is too low to be detected, either visually or with instruments.
To amplify the difference, the prior art use the time consuming cell culture to replicate bacteria. This invention, however, amplifies the difference by in vitro DNA replication, e.g. in vitro amplification of the common 16S rRNA gene among bacteria.
DNA amplification methods such as polymerase chain reaction (PCR), strand displacement amplification, and self-sustained sequence replication have been used to replicate 16S rRNA gene specific to different bacteria for genera or species identification. (See for examples, "Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction" Wilson et al., J. of Clin. Microbiol., 28(9), 1942-1946, 1990; "Strand Displacement Amplification - an Isothermal, in vitro DNA amplification techniques" Walker et al., Nucleic Acids Res., 20(7), 1601-1696, 1992; and "Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication" Guatelli et al., Proc. Natl. Acad. Sci. USA, 87, 1874-1878, 1990.) In addition, PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria have been reported in Greisen et al., J. of Clin. Microbiol., 32(2), 335-350, 1994.
This invention, however, teaches new applications of these in vitro amplification methods to replicate conserved region of 16S rRNA gene for antimicrobial susceptibility testing.
To expose the bacterial genetic material for amplification, the microorganisms are lysed with lysing reagents. Suitable lysing agents for exposing genomic DNA or RNA for amplification include, but not limited to, lysozyme and proteinase K.
To amplify bacterial DNA, the prior art require that the bacteria be purified from culture medium before the lytic reagents are applied. After bacterial cells are lysed, the lytic reagents must be removed before the DNA amplification can proceed because the amplification process requires DNA polymerases that are vulnerable to lytic reagents. However, to purify a large number of samples in microtiter plate format is not practical and is time consuming. This invention teaches that diluting the culture medium or the lytic reagents by 10-fold is sufficient to reduce the various inhibiting components to levels that do not interfere with the amplification process.
Therefore, after exposing genetic material of microorganisms, amplification can be performed directly without removing ingredients, e.g. lytic agents, in the culture medium by centrifugation or other purification processes.
The in vitro DNA amplification methods achieve one round of DNA replication in less than three minutes. In comparison, for fast growing bacterium like E. coli, the traditional cell culture method requires 20 minutes for one round of replication and approximately 20 hours for slow growing bacterium like M. tuberculosis.
DNA amplification can be conducted either by polymerase chain reaction for preferably less than 30, e.g. 25 cycles, in a multi-well PCR machine, or by other DNA replication methods, e.g. isothermal in vitro amplification, for less than four hours, preferably less than three and most preferably less than two hours.
To analyze DNA molecules amplified by PCR or other DNA amplification methods, electrophoresis is the conventional method of choice. By identifying a band on a slab gel one can determine whether the DNA amplification reaction has generated the desired product. This analytical method is acceptable when only a few samples are analyzed. In antimicrobial susceptibility testing or in other screening methods where large number of samples are employed on a microtiter plate, using electrophoresis as the analytical tool is intolerably tedious and impractical. This invention recognizes the limitations which arise from a large number of DNA amplification product analysis and provides a quick solution to solve the problem.
There are many ways to distinguish dsDNA molecules from free deoxynucleotides by their physical properties such as absorbance (hypochromacity) or circular dichroism. (See e.g. D. Freifelder, Physical Biochemistry, 2nd Ed. W. H. Freeman & Co.) However, drawing samples one by one and measuring them spectrophotometrically does not improve in detection speed over that of gel electrophoresis. Newer PCR product analyzing methods utilizing electro-chemiluminescence principles or enzyme colorimetric detection principles to simultaneously detect large number of PCR products are available. (See e.g. Miller, L. A. et al., J. Clin Microbiol. 32(5), 1373-1375, 1994; and Roy, Y. R. et al., J. Clin. Microbiol. 22(3), 467-469, 1995.) However, these methods require blocking, incubation, and washing procedures which can delay the process of susceptibility testing.
In accordance with the invention, reporter molecules which are specific to dsDNA and form complexes therewith are utilized to distinguish the dsDNA generated by DNA replication methods from the free nucleotides dominating in the wells that contain efficacious antimicrobial agent. The signal emission from the complexes provides an efficient means for an immediate visual determination of which wells have gone through DNA amplification to contain high bacterial DNA concentrations.
Some fluorescent intercalating dyes selectively bind to dsDNA and give out enhanced fluorescence emission when bound. Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection have been reported in Glazer, A. N., Rye, H. S., Nature, 359, 859-861, 1992. The intercalating dyes of choice are, for example, 1, 1'-(4,4,7,7,-tetramethyl-4,7-diazaundecamethylene)-bid-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazo)-2-methyl-idene]-quinolinium tetraiodide, designated TOTO or 1,1'-(4,4,7,7-tetra-methyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinolinium tetraiodide, designated YOYO. These dyes allow much more sensitive fluorescence detection of double stranded DNA than previously possible.
The fluorescence emission enhancement of YOYO or TOTO when bound to dsDNA are on the order of thousands, the F_bound vs. F_free is 3200 for YOYO and 1100 for TOTO. Compared to ethidium bromide (F_bound/F_free ∼30) which is the most widely used intercalating dye for detecting dsDNA, YOYO or TOTO offers much higher signal-to-noise ratio in detection. Therefore, by using intercalating dye with very high fluorescence enhancement ratio, one can distinguish, by fluorescence intensity, wells that contain dsDNA from those that do not.
Intercalating dyes such as propidium iodide or ethidium homodimer can also be used for rapid detection of DNA amplification. Because ethidium bromide has larger absorptivity in the UV range than YOYO, ethidium bromide appears to have brighter fluorescence emission than YOYO when UV transillumination light box is used as the excitation source. However, ethidium bromide has much lower F_bound vs. F_free ratio than YOYO. Therefore, the background emission of free ethidium bromide is significantly higher than free YOYO so that the dynamic range and signal-to-noise ratio of detection are less with ethidium bromide. This lower UV absorptivity of YOYO can be overcome by using blue light wavelengths ranging between 450-490 nm to excite the dye. The light source can either be a high power xenon lamp or tungsten halogen lamp with a bandpass filter optimized to pass excitation wavelengths from 450 nm to 490 nm or a laser with output ranging within that particular wavelength region, for instance, an argon ion laser with 488 nm output.
To generate and measure fluorescence emission from individual well, a laser induced fluorescence microtiter plate reader can be used. By using laser induced fluorescence detection, the fluorescence emissions from the micro-wells are sufficiently intense such that minimal signal integration time is required and a microtiter plate containing 96 samples can be read in less than 20 seconds.
The detection method in accordance with the invention using intercalating dye is rather simple. After the amplification reaction is done, the dye such as ethidium bromide can be pipetted in the container, e.g. a microtiter plate, and the container is placed over a UV trans-illumination light box. The wells that contain amplified dsDNA will lit up brightly while the other wells remain dim. Using either of these powerful intercalating dyes, 96 amplified samples can be detected on a microtiter plate in just a few seconds by observing the plate with one's eyes.
Alternatively, the microtiter plate can be read by a fluorescence microtiter plate reader with Xenon arc lamp as the excitation source. The intensity of the fluorescence or the concentration of the bacteria in the wells can be quantitated with appropriate fluorescence bandpass filters.
To have quantitative data, fluorescent intercalating dyes such as ethidium bromide, SYBR I, or YOYO is added to each well either before or after the amplification reaction. Fluorescence intensities from the wells that contain efficacious antimicrobial agents are low due to few or no replication templates while the wells that contain inefficacious antimicrobial agents yield high fluorescence intensities.
For quantitative purpose, controls in two microtiter wells are needed. The signal arising from the well containing no antimicrobial agent but culture medium alone represents full scale growth with no inhibition. The signal arising from the well containing lytic agents such as lysozyme represents zero growth. To assure good linear correlation between fluorescence intensity and bacterial counts in wells, quantitative DNA amplification must be performed within a linear range, which means sample dilution and a low number of amplification cycles must be employed.
To report results, the lowest antimicrobial concentration to have the same fluorescence intensity as the zero growth control is recorded as the Minimum Inhibition Concentration (MIC).
The entire process, from the placement of specimens in microtiter wells to the retrieval of MIC values, can be completed in less than 6 hours. Compared to the traditional broth dilution or disk diffusion methods which require visual detection of bacterial colonies, this novel system is 10-fold faster. Furthermore, this system is applicable to antimicrobial susceptibility testing for fungal or mycobacterial infections. The results of susceptibility testing can be obtained in one day for fungi and less than 10 days, compared to the routine 6 to 8 weeks as required by prior art, for mycobacteria.
Taken altogether, this innovative system is an integrated approach to identify efficacious antimicrobial agents which save lives of many critically ill bacteremia patients. Particularly at the time when drugs which effectively control sepsis are yet to be discovered, eradicating pathogens in good time will prevent patients from developing septic shock and increase the odds of survival from the infection.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way.

EXAMPLE 1

To demonstrate and realize the invention, Escherichia coli ATCC 25922 as recommended by NCCLS (National Committee for Clinical Laboratory Standards) was used. The strain is genetically stable and has tabulated MIC ranges published by NCCLS as follows for ampicillin, cephalothin, nalidixic acid and gentamicin.

Quality control MIC range E. coli ATCC 25922

Antibiotics MIC range µg/ml)

Ampicillin 2.0-8.0

Cephalothin 4.0-16.0

Nalidixic Acid 1.0-4.0

Gentamicin 0.25-1.0
One hundred µl of E. coli, ATCC 25922, inoculum with concentrations approximately 2.5 x 10⁴ CFU/ml in Mueller-Hinton broth was inoculated in each well of a 96-well microtiter plate. The stock solutions of the antibiotics were prepared according to NCCLS recommendations. Appropriate concentrations of each kind of the following antimicrobial agent was added to the first row of the microtiter plate. Serial 2-fold dilutions of the antimicrobial agents being tested was performed in each row to generate eight rows of varying antimicrobial concentrations: ampicillin (0.25∼32 µg/ml); cephalothin (0.5∼32 µg/ml); gentamicin (0.125∼16 µg/ml; and nalidixic acid (0.25∼32 µg/ml).
Following a 3-hour incubation interval at 37°C, 3 µl amount of each well sample was transferred to a V-bottom autoclavable microtiter plate for further treatment.
To lyse bacterial cells in each microtiter well and to expose the DNA, lysozyme and lysostaphin were added to the wells at final concentrations of 1 mg/ml and 100 µg/ml, respectively. The samples were incubated at 37°C for 30 minutes prior to the addition of proteinase K and Tween 20 to final concentrations of 100 µg/ml and 1%(V/V), respectively. An additional 30 minutes of incubation at 55°C was allowed to complete the lysis procedures before the enzymes were inactivated at 95°C for 10 minutes.
Bacterial DNA molecules were amplified in 40 µl PCR mixture consisting of 4 µl of 10 x buffer solution (100 mM Tris-HCl, pH 8.8, 1.5 mM MgCl₂, 50 mM KCl, and 0.1% Triton X-100), 0.125 µl each of 20 µM PCR primers with nucleotide sequences 5'-AGGAGGTGATCCAACCGCA and 5'-AACTGGACCAAGGTGGGGAG, 0.2 µl of 10 mM dNTP mixture, 12 µl of bacterial lysate, 0.5 µ of 2U/µl polymerase (Dynazyme), and 23.1 µl of H₂O. Additional 15 µl of mineral oil was added to prevent evaporation. The microtiter plate containing the PCR mixtures was placed in a 96-well block thermal cycler (PTC-100, MJ Research, Inc.). The thermal cycler was set to 94°C for 7 minutes followed by 25 cycles of the following temperature profiles, 95°C for 45 seconds, 55°C for 30 seconds, 72°C for 40 seconds, and a final extension at 72°C for 5 minutes.
After DNA amplification, the products were transferred to a flat bottom 96-well microtiter plate and YOYO-1 dye (Molecular Probes, Eugene, OR) in 0.1 x TAE buffer solution was added to the wells to make final dye concentration of 0.5 µM. The concentrations of amplified DNA were measured in less than one minute by an in-house constructed laser induced fluorescence microtiter plate reader which was consisted of an argon ion laser, photomultiplier tube, detection electronic circuits, and a computer controlled X-Y translation stage. The fluorescence image acquired by the scanning system was pseudo-color encoded for easy identification of differences in fluorescence intensity.
The results are shown in Fig. 3. The MIC values reported by this invention agree well with the above tabulated MIC values published by NCCLS.

EXAMPLE 2

With a similar process, the MIC values for various antimicrobial agents to reference bacterial strains Pseudomonas aerugenosa ATCC 27853, staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 were determined. The MIC values for a panel of antibiotics were read under UV illumination in just a few seconds by observing the plates with or without the aid of a CCD camera and integrator. In such embodiment, the intercalating dye added to the wells has absorption within the UV region. For instance, ethidium bromide was added to the wells to final concentration of 3µM and the microtiter plate was placed on top of a UV light box that output 313 nm wavelength UV light or be illuminated by UV light from above. The fluorescence emission of the ethidium bromide intercalated DNA can be viewed through a bandpass filter with center wavelength around 610 nm.

The susceptibility testing results of various antimicrobial agents to the three bacterial strains by this invention, the in vitro DNA amplification and fluorescence emission (DAFE) method, are summarized in the following table.

Antimicrobial agent	Bacterial Strain	MIC range (µg/ml)
		NCCLS	DAFE
Ampicillin		2-8	4-16
Carbenicillin		4-16	16-32
Cephalothin		4-16	4-32
Cefuroxime	E. coli	2-8	4
Cefotaxime	(ATCC 25922)	0.06-0.25	1
Ceftriaxone		0.03-0.12	0.06-0.12
Gentamicin		0.25-1	0.5
Norfloxacin		0.03-0.12	0.03-0.06
Nalidixic Acid		1-4	2-8
Cephalothin		0.12-0.5	0.12-0.5
Clindamicin		0.06-0.25	0.06
Erythromicin		0.12-0.5	0.06-0.12
Gentamicin	S. aureus	0.12-1	0.12-0.25
Norfloxacin	(ATCC 29213)	0.5-2	1-2
Penicillin G		0.25-1	0.25-1
Rifampin		0.008-0.06	0.004-0.015
Trimethoprim		1-4	1-2
Vancomycin		0.5-2	1
Carbenicillin		16-64	16-64
Ceftriaxone	P. aeruginosa	8-32	8-32
Gentamicin	(ATCC 27853)	1-4	0.25-1

Claims

A method for rapid antimicrobial susceptibility testing which comprises:

(a) incubating a specimen comprising one or more microbes, in a medium comprising an antimicrobial agent at serial dilution concentrations for from 1 to 6 hours to create differential microbial counts;

(b) amplifying the differential microbial counts by in vitro microbial DNA replication;

(c) contacting the amplified differential counts with one or more reporter molecules which are specific to double stranded DNA (dsDNA) and form complexes therewith; and

(d) observing the presence or absence of signal emission from the reporter molecule-dsDNA complexes.
A method for rapid determination of minimum inhibitory concentration (MIC) values of antimicrobial agents which comprises:

(a) incubating specimens comprising one or more microbes in a medium comprising an antimicrobial agent at serial dilution concentrations for from 1 to 6 hours to create differential microbial counts;

(b) amplifying the differential microbial counts by in vitro microbial DNA replication;

(c) contacting the amplified differential counts with one or more reporter molecules which are specific to dsDNA and form complexes therewith; and

(d) recording the lowest concentration of antimicrobial agent which does not result in signal emission from the reporter molecule-dsDNA complexes as the MIC value.
A method according to claim 1 or 2, for antimicrobial susceptibility testing of a prokaryotic organism.
A method for rapid antimicrobial susceptibility testing for slow-growing microorganisms which comprises:

(a) incubating a specimen comprising one or more slow-growing microorganisms in a medium comprising an antimicrobial agent at serial dilution concentrations for less than 24 hours to create differential microbial counts or, when the slow growing microorganism is Mycobacterium tuberculosis, for less than 10 days;

(b) amplifying the differential microbial counts by in vitro microbial DNA replication;

(c) contacting the amplified differential counts with one or more reporter molecules which are specific to dsDNA and form complexes therewith; and

(d) observing the presence or absence of signal emission from the reporter molecule-dsDNA complexes.
A method according to claim 4 for antimicrobial susceptibility testing of fungi or Mycobacteria.
A method according to any one of the preceding claims wherein no primary culture of the specimen is involved.
A method according to any one of the preceding claims wherein no prior pathogen identification of the microbe or microbes comprised in the specimen is involved.
A method according to any one of the preceding claims wherein the microbes or microorganisms are lysed and their DNA exposed in the medium without removing the ingredients of the medium.
A method according to claim 8 wherein the DNA is amplified without removing the lytic reagents.
A method according to claim 8 or 9 wherein the method is a means to reduce the concentration of inhibiting components.
A method according to any one of the preceding claims wherein the microbial concentration of the specimen is below 10⁵ CFU/ml.
A method according to any one of the preceding claims wherein the DNA amplification is performed by a thermal cycling amplification method.
A method according to claim 12, wherein the thermal cycling amplification method is a polymerase chain reaction (PCR).
A method according to claim 13 wherein the PCR comprises less than 30 cycles.
A method according to any one of the preceding claims 1 to 11 wherein the DNA amplification is performed by an isothermal amplification method.
A method according to claim 15, wherein the isothermal amplification method is strand displacement amplification or self-sustained sequence replication (3SR).
A method according to any one of the preceding claims wherein the DNA amplification is of a 16S rRNA target DNA.
A method according to any one of the preceding claims wherein the DNA amplification is performed in a multi-well plate.
A method according to any one of the preceding claims wherein the reporter molecule is a fluorescent intercalating dye.
A method according to claim 19 wherein the fluorescence is emitted from the intercalating fluorescent dye complexed with the dsDNA and the fluorescence emission is stronger than the intercalating fluorescent dye in its non-intercalated form.
A method according to claim 19 or 20 wherein fluorescence is induced by UV excitation or laser excitation.
A method according to claim 21 wherein the UV excitation is at a wavelength of from 250 nm to 400 nm.
A method according to claim 19 or 20 wherein the fluorescent intercalating dye comprises a phenanthridinium monomer.
A methods according to claim 22 wherein the fluorescent intercalating dye is ethidium bromide.
A method according to claim 19, wherein fluorescence is induced by a laser excitation.
A method according to any one of claims 19 to 25 wherein the fluorescent intercalating dye is a multimer.
A method according to claim 26, wherein the fluorescent intercalating dye comprises a phenanthridine subunit.
A method according to claim 27, wherein the fluorescent intercalating dye is a dimer comprising an acridine subunit.
A method according to claim 28 wherein the MIC values of the antimicrobial agent against the microbe or microbes is determined by observation by eye or by an electronic device.
A method according to claim 29 wherein the electronic device comprises a photomultiplier tube detector and a computer.
A method according to any one of the preceding claims comprising quantifying the signal emission from the reporter molecule-dsDNA complexes.
A method according to any one of the preceding claims wherein the specimen is a human specimen.